A purification method of natural antibodies derived from alpacas in South America and its applications

Abstract

The present invention discloses a purification method and application of natural antibodies from alpacas in America. The purification method of the natural antibodies includes the following steps: S1, pretreatment of alpaca serum in America; S2, purification by protein A column; S3, ion exchange chromatography; S4, hydroxyapatite chromatography. Through specific purification steps (firstly, pretreatment of alpaca serum in America, then purification by protein A column, then ion exchange chromatography, and finally hydroxyapatite chromatography), and there is a synergistic effect among the above purification steps, natural antibodies IgG1, natural heavy chain antibodies IgG2 and natural heavy chain antibodies IgG3 with extremely high purity and yield are finally obtained. The natural antibodies have good application prospects in the technical field of preparing antibodies against natural antibodies.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to a purification method and application of natural antibodies derived from alpacas in America. Background Art

[0002] Immunoglobulin (Ig) refers to a globulin that has antibody activity or a chemical structure similar to that of an antibody and plays a key role in the human immune response process. Different types of immunoglobulins generally have the same basic structure, usually including four peptide chains, namely two identical heavy chains and two identical light chains, which are connected by disulfide bonds. The molecular weight of the heavy chain can reach 50 - 75 kDa, while the molecular weight of the light chain is very small, about 25 kDa. According to the amino acid composition and arrangement order of the constant region of the heavy chain, immunoglobulins can be divided into five categories, namely IgG, IgE, IgD, IgA, and IgM. According to the amino acid composition and arrangement order of the constant region of the light chain, the light chain can be further divided into kappa light chain and lambda chain. However, on the same immunoglobulin, the two light chains should be of the same type.

[0003] At the position of 110 amino acids near the N-terminus of the immunoglobulin heavy and light chains, the amino acid sequence varies greatly, and this region is called the variable region (V H and V L ). The variable region can provide antigen-binding sites with high affinity and high specificity. Most of these antigen-binding sites are between the amino acid residues in the complementarity-determining region and the main epitope of the antigen. Therefore, the basic function of the variable region of immunoglobulin is to recognize and specifically bind antigens. At the same time, the variable region also plays an important role in the binding of immunoglobulins to bacterial toxins and viruses, preventing the invasion of pathogens. The amino acid sequence near the C-terminus of the immunoglobulin heavy and light chains varies less, and this region is called the constant region (C H and C L ). The most important functions of the constant region are as follows: 1) activating complement; 2) binding to Fc receptor molecules (FcR) and mediating various biological effects; 3) positive and negative regulatory effects on the immune response. The Fc segment binds to FcR on neutrophils and macrophages, which can enhance the phagocytic function of phagocytes; while cytotoxic cells can recognize and bind to the Fc segment of antibodies bound to target antigens through their Fc receptors, thereby directly killing cells. This process is called antibody-dependent cell-mediated cytotoxicity (ADCC).

[0004] In 1993, Hamers Casterman in Belgium (Hamers-Casterman C, Atarhouch T, Muyldermans S, et al. Naturally occurring antibodies devoid of light chains. [J]. Nature, 1993, 363(6428):446.) reported that in camel serum, there not only existed conventional IgG1 antibodies composed of 2 light chains and 2 heavy chains, but also heavy-chain antibodies (hcAb) of IgG2 and IgG3 subtypes lacking light chains, which had complete antigen-binding ability. And IgG antibodies were purified from camel serum. Among them, IgG1 was composed of a 50 kDa heavy chain and a 30 kDa light chain, IgG2 was only composed of a 46 kDa heavy chain, and IgG3 was only composed of a 43 kDa heavy chain. Currently, studies have shown that IgG1 and IgG3 bind to protein G and protein A, while IgG2 only binds to protein A. Therefore, only IgG2 can be obtained by affinity chromatography, and IgG1 and IgG3 still cannot be separated, and the purity and yield after separation are relatively low. Therefore, it is necessary to provide a method for separating and purifying high-purity heavy-chain antibodies from alpaca serum. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a purification method and application of natural antibodies derived from alpacas. It solves the problems that only IgG2 can be obtained by affinity chromatography currently, IgG1 and IgG3 still cannot be separated, and the purity and yield after separation are relatively low.

[0006] To solve the above problems existing in the prior art, the present invention is realized through the following technical solutions:

[0007] A purification method of natural antibodies derived from alpacas, comprising the following steps:

[0008] S1. Pretreatment of alpaca serum: Using the caprylic acid-ammonium sulfate precipitation method, extract the natural antibodies in alpaca serum to obtain a pretreatment solution;

[0009] S2. Purification by protein A column: Use a protein A column to purify the pretreatment solution obtained in step S1 to obtain natural heavy-chain antibody IgG2 and a primary treatment solution;

[0010] S3. Ion exchange chromatography: Use an anion exchange chromatography column to purify the primary treatment solution obtained in step S2 to obtain natural antibody IgG1 and an intermediate treatment solution;

[0011] S4, Hydroxyapatite chromatography: Using a hydroxyapatite chromatography column, purify the intermediate treatment solution obtained in step S3 to obtain natural heavy chain antibody IgG3 and a small amount of natural heavy chain antibody IgG2.

[0012] Furthermore, in step S1, the specific operation steps of the caprylic acid - saturated ammonium sulfate precipitation method are as follows: Mix alpaca serum with an acetate buffer solution with a pH of 4 - 6 at a volume ratio of 1:(2 - 3), add 3 - 5% caprylic acid, mix at room temperature, then let it stand overnight at 2 - 8°C to fully precipitate; Centrifuge to obtain the supernatant, add an equal volume of saturated ammonium sulfate solution, mix well, let it stand overnight at 2 - 8°C, centrifuge to obtain the precipitate, and extract with 3 - 5 times the weight of the precipitate of saturated ammonium sulfate solution for 3 - 5 times. The last precipitate is dissolved in PBS buffer solution, and after dialysis, the pretreatment solution can be obtained.

[0013] Even further, the alpaca serum needs to be pretreated, and the specific method is as follows: Centrifuge at 7000 - 9000 r / min for 10 - 20 min, collect the supernatant, which is the pretreated alpaca serum.

[0014] Furthermore, step S2 specifically includes the following steps:

[0015] S21, Loading: First, equilibrate the protein A column with 10 - 12 column volumes of PBS buffer solution, then load the pretreatment solution obtained in step S1 into the protein A column, and wash it with 5 - 8 column volumes of 10 mM PBS buffer solution with a pH of 7.0 - 7.2 at a flow rate of 1 - 2 mL / min to obtain the primary treatment solution;

[0016] S22, Elution: Continue to wash the protein A column in step S21 with 3 - 5 column volumes of 20 mM citric acid buffer solution with a pH of 4.0 - 5.0 at a flow rate of 0.5 - 1 mL / min, collect the eluate, and add 1 M Tris - HCl buffer solution with a pH of 8.5 to adjust the solution pH to 7.2 - 7.4, which is the natural heavy chain antibody IgG2.

[0017] Even further, before loading, it also includes the step of sample treatment, specifically as follows: Mix the pretreatment solution obtained in step S1 with an equal volume of 10 mM PBS buffer solution with a pH of 7.0 - 7.2, and then filter it through a 0.45 μm filter membrane to obtain the sample to be purified.

[0018] Furthermore, in step S3, the packing material of the anion - exchange chromatography column is selected from one of DEAE - Sepharose, Capto - DEAE, Q Sepharose, Capto - Q, and QAE Sephadex.

[0019] Further, step S3 specifically includes the following steps:

[0020] S31. Loading: First, equilibrate the anion exchange chromatography column with 8 - 10 column volumes of PBS buffer. Then, load the primary treatment solution obtained in step S2 onto the anion exchange chromatography column, and rinse it with 4 - 6 column volumes of 10 mM PBS buffer with a pH of 7.0 - 7.2 at a flow rate of 1 - 2 mL / min to obtain an intermediate treatment solution.

[0021] S32. Elution: Continuously rinse the anion exchange chromatography column in step S31 with 3 - 6 column volumes of 50 mM Tris - HCl buffer (containing 150 - 300 mM NaCl) with a pH of 7.2 - 8.0 at a flow rate of 0.5 - 0.8 mL / min. Collect the eluate and add 1 M Tris - HCl buffer with a pH of 8.5 to adjust the solution pH to 7.2 - 7.4, which is the natural antibody IgG1.

[0022] Further, in step S4, the hydroxyapatite chromatography column is a ceramic hydroxyapatite chromatography column (CHT TypeⅡ).

[0023] Further, step S4 specifically includes the following steps:

[0024] S41. Loading: First, equilibrate the hydroxyapatite chromatography column with 6 - 8 column volumes of PBS buffer. Then, load the intermediate treatment solution obtained in step S3 onto the hydroxyapatite chromatography column, and rinse it with 4 - 6 column volumes of 10 mM PBS buffer with a pH of 7.0 - 7.2 at a flow rate of 1 - 2 mL / min. Collect the effluent and add 1 M Tris - HCl buffer with a pH of 8.5 to adjust the solution pH to 7.2 - 7.4, which is a small amount of natural heavy - chain antibody IgG2.

[0025] S42. Elution: Continuously rinse the anion exchange chromatography column in step S41 with 4 - 6 column volumes of 100 mM glycine buffer with a pH of 2.0 - 3.0 at a flow rate of 0.3 - 0.5 mL / min. Collect the eluate and add 1 M Tris - HCl buffer with a pH of 8.5 to adjust the solution pH to 7.2 - 7.4, which is the natural heavy - chain antibody IgG3.

[0026] The present invention also provides an application of the natural antibody obtained by the purification method of the natural antibody derived from alpacas in the preparation of an antibody against the natural antibody as described in any one of the above.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] 1) The present invention first purifies the serum by the n - octanoic acid - saturated ammonium sulfate precipitation method to obtain a pretreatment solution, removing impurities while concentrating the natural antibody to be purified; then it uses a protein A column for purification. Since the protein A column can specifically bind IgG2, thus, a natural heavy - chain antibody IgG2 and a primary treatment solution are further obtained. Then, the above - mentioned primary treatment solution is purified using an anion - exchange chromatography column to obtain a natural antibody IgG1 and an intermediate treatment solution; because ceramic hydroxyapatite has good capture ability and selectivity, and can separate various difficult - to - separate and property - similar biological molecules that other media cannot separate, therefore, using ceramic hydroxyapatite can greatly reduce the purification steps and the resulting product has a high purity. In the present invention, finally, the intermediate treatment solution is purified using a hydroxyapatite chromatography column, which can separate a small amount of natural heavy - chain antibody IgG2 and natural heavy - chain antibody IgG3 with close molecular weights in the intermediate treatment solution, obtaining a higher - purity IgG3 while also improving the yield and purity of IgG2;

[0029] 2) The present invention passes through specific purification steps (firstly, pre - treating the serum of alpacas, then purifying with a protein A column, then performing ion - exchange chromatography, and finally performing hydroxyapatite chromatography), and there is a synergistic effect among the above - mentioned purification steps. Finally, a natural antibody IgG1, a natural heavy - chain antibody IgG2, and a natural heavy - chain antibody IgG3 with extremely high purity and yield are obtained. The natural antibody has good application prospects in the technical field of preparing antibodies against natural antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following - described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0031] Figure 1 It is the SDS - PAGE electrophoresis diagram in Embodiment 1 of the present invention; among them, lane M: protein Marker, lane 1: purified natural antibody IgG1; lane 2: purified natural heavy - chain antibody IgG2, lane 3: purified natural heavy - chain antibody IgG3. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0033] Conventional reagents and equipment used in the present invention can be commercially obtained without special instructions.

[0034] Example 1 Purification of Natural Antibodies in Alpacas

[0035] A purification method for natural antibodies derived from alpacas includes the following steps:

[0036] S1. Pretreatment of alpaca serum: First, pretreat the alpaca serum. The specific method is as follows: Centrifuge the alpaca serum at 7000 - 9000 r / min for 10 - 20 min, and collect the supernatant, which is the pretreated alpaca serum; then mix the pretreated alpaca serum with an acetate buffer solution with a pH of 4 - 6 at a volume ratio of 1:(2 - 3), add 3 - 5% n-octanoic acid, mix at room temperature, and then let it stand overnight at 2 - 8°C to fully precipitate; centrifuge to obtain the supernatant, add an equal volume of saturated ammonium sulfate solution, mix well, let it stand overnight at 2 - 8°C, centrifuge to obtain the precipitate, and extract the precipitate with 3 - 5 times the weight of the precipitate of saturated ammonium sulfate solution for 3 - 5 times. The last precipitate is dissolved in PBS buffer solution, and after dialysis, the pretreatment solution can be obtained;

[0037] S2. Protein A column purification: First, mix the pretreatment solution obtained in step S1 with an equal volume of 10 mM PBS buffer solution with a pH of 7.0 - 7.2, and then filter through a 0.45 μm filter membrane to obtain the sample to be purified; then perform sample loading: First, equilibrate the protein A column with 10 - 12 column volumes of PBS buffer solution, and then load the pretreatment solution obtained in step S1 onto the protein A column, and wash it with 5 - 8 column volumes of 10 mM PBS buffer solution with a pH of 7.0 - 7.2 at a flow rate of 1 - 2 mL / min to obtain the primary treatment solution; finally, perform elution: Continue to wash the protein A column with 3 - 5 column volumes of 20 mM citrate buffer solution with a pH of 4.0 - 5.0 at a flow rate of 0.5 - 1 mL / min, collect the eluate, and add 1 M Tris-HCl buffer solution with a pH of 8.5 to adjust the pH of the solution to 7.2 - 7.4, which is the natural heavy chain antibody IgG2;

[0038] S3. Ion exchange chromatography: Use an anion exchange chromatography column (DEAE-Sepharose, Capto-DEAE, Q Sepharose, Capto-Q, QAE Sephadex) for sample loading. First, equilibrate the anion exchange chromatography column with 8 - 10 column volumes of PBS buffer. Then, load the primary treatment solution obtained in step S2 onto the anion exchange chromatography column and wash it with 4 - 6 column volumes of 10 mM PBS buffer with a pH of 7.0 - 7.2 at a flow rate of 1 - 2 mL / min to obtain an intermediate treatment solution. Then, perform elution: Continue to wash the anion exchange chromatography column with 3 - 6 column volumes of 50 mM Tris-HCl buffer (containing 150 - 300 mM NaCl) with a pH of 7.2 - 8.0 at a flow rate of 0.5 - 0.8 mL / min. Collect the eluate and add 1 M Tris-HCl buffer with a pH of 8.5 to adjust the solution pH to 7.2 - 7.4, which is the natural antibody IgG1.

[0039] S4. Hydroxyapatite chromatography: Use a ceramic hydroxyapatite chromatography column (CHT TypeⅡ) for sample loading. First, equilibrate the hydroxyapatite chromatography column with 6 - 8 column volumes of PBS buffer. Then, load the intermediate treatment solution obtained in step S3 onto the hydroxyapatite chromatography column and wash it with 4 - 6 column volumes of 10 mM PBS buffer with a pH of 7.0 - 7.2 at a flow rate of 1 - 2 mL / min. Collect the effluent and add 1 M Tris-HCl buffer with a pH of 8.5 to adjust the solution pH to 7.2 - 7.4, which is a small amount of natural heavy chain antibody IgG2. Then, perform elution: Continue to wash the anion exchange chromatography column in step S41 with 4 - 6 column volumes of 100 mM glycine buffer with a pH of 2.0 - 3.0 at a flow rate of 0.3 - 0.5 mL / min. Collect the eluate and add 1 M Tris-HCl buffer with a pH of 8.5 to adjust the solution pH to 7.2 - 7.4, which is the natural heavy chain antibody IgG3.

[0040] Perform SDS-PAGE electrophoresis on the llama serum, the sample after step S1 treatment, the sample after step S2 treatment, the sample after step S3 treatment, and the sample after step S4 treatment. The results are as Figure 1 shown.

[0041] Comparative Example 1

[0042] The purification method of natural antibodies in llamas is basically the same as that in Example 1, except that the pretreatment step of llama serum is lacking.

[0043] Comparative Example 2

[0044] The method for purifying natural antibodies in alpacas is basically the same as that in Example 1, except that the protein A column purification step is missing.

[0045] Comparative Example 3

[0046] The method for purifying natural antibodies in alpacas is basically the same as that in Example 1, except that the ion exchange chromatography step is missing.

[0047] Comparative Example 4

[0048] The method for purifying natural antibodies in alpacas is basically the same as that in Example 1, except that the hydroxyapatite chromatography step is missing.

[0049] Comparative Example 5

[0050] The method for purifying natural antibodies in alpacas is basically the same as that in Example 1, except that the hydroxyapatite chromatography step is located between the pretreatment step of alpaca serum and the protein A column purification step, and other steps remain unchanged.

[0051] Comparative Example 6

[0052] The method for purifying natural antibodies in alpacas is basically the same as that in Example 1, except that the hydroxyapatite chromatography step is located between the protein A column purification step and the ion exchange chromatography step, and other steps remain unchanged.

[0053] Performance test on the yield and purity of natural antibodies in Example 2

[0054] The natural antibodies prepared in Example 1 and Comparative Examples 1-6 were subjected to yield and purity performance tests, and the results are shown in Table 1 below:

[0055] Table 1 Results of yield and purity performance tests on natural antibodies

[0056]

[0057] It can be seen from the results in the table that the natural antibodies obtained in Example 1 have extremely high purity, all above 95%, and the yield is also above 80%. The results show that through the specific purification steps of the present invention, natural antibodies IgG1, natural heavy chain antibodies IgG2 and natural heavy chain antibodies IgG3 with extremely high purity and yield are obtained;

[0058] The differences between Comparative Examples 1 to 4 and Example 1 lie in the absence of one of the steps of pretreatment of alpaca serum, purification by Protein A column, ion exchange chromatography, and hydroxyapatite chromatography. It was found that the purity and yield of the obtained natural antibodies IgG1, natural heavy-chain antibody IgG2, and natural heavy-chain antibody IgG3 all decreased to a certain extent. The results indicate that there is a synergistic effect among the above purification steps. When a certain step is missing, the purity and yield of the purified natural antibodies both decrease to a certain extent;

[0059] The differences between Comparative Example 5 and Comparative Example 6 and Example 1 are that the hydroxyapatite chromatography step is located between the pretreatment step of alpaca serum and the Protein A column purification step, or the hydroxyapatite chromatography step is located between the Protein A column purification step and the ion exchange chromatography step, and other steps remain unchanged. It was found that the purity and yield of the obtained natural antibodies IgG1, natural heavy-chain antibody IgG2, and natural heavy-chain antibody IgG3 all decreased to a certain extent, and the decrease in the purity of heavy-chain antibody IgG2 in Comparative Example 5 was more obvious. This is because when hydroxyapatite chromatography is carried out first, due to the simultaneous presence of 3 heavy-chain antibodies and the limited capacity of the hydroxyapatite chromatography column, there is a loss of heavy-chain antibodies during separation, resulting in a low purity and yield of the obtained heavy-chain antibodies. The results indicate that when the hydroxyapatite chromatography step is located at the last step, since the sample only contains natural heavy-chain antibody IgG3 and a small amount of natural heavy-chain antibody IgG2, and the types of samples to be separated are few, therefore, a natural antibody with extremely high purity can be obtained by purification with hydroxyapatite chromatography.

[0060] In summary, the present invention fully considers the properties of the natural antibodies to be separated, and through specific purification steps (firstly, pretreating alpaca serum, then purifying by Protein A column, then performing ion exchange chromatography, and finally performing hydroxyapatite chromatography), and there is a synergistic effect among the above purification steps, and finally natural antibodies IgG1, natural heavy-chain antibody IgG2, and natural heavy-chain antibody IgG3 with extremely high purity and yield are obtained.

[0061] The above embodiments are only specific embodiments of the present invention, used to illustrate the technical solutions of the present invention, rather than limiting it. The protection scope of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: Any person skilled in the art within the technical scope disclosed by the present invention can still modify the technical solutions recorded in the foregoing embodiments, or can easily think of changes, or perform equivalent replacements on some of the technical features; and these modifications, changes or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be covered within the protection scope of the present invention.

Claims

1. A purification method for natural antibodies derived from alpacas in America, characterized in that, It includes the following steps: S1. Pretreatment of alpaca serum: Using the caprylic acid-ammonium sulfate precipitation method, extract the natural antibodies in the alpaca serum to obtain a pretreatment solution; S2. Purification by protein A column: Use a protein A column to purify the pretreatment solution obtained in step S1 to obtain natural heavy chain antibody IgG2 and a primary treatment solution; S3. Ion exchange chromatography: Use an anion exchange chromatography column to purify the primary treatment solution obtained in step S2 to obtain natural antibody IgG1 and an intermediate treatment solution; S4. Hydroxyapatite chromatography: Use a hydroxyapatite chromatography column to purify the intermediate treatment solution obtained in step S3 to obtain natural heavy chain antibody IgG3 and a small amount of natural heavy chain antibody IgG2; In step S1, the specific operation steps of the caprylic acid-ammonium sulfate precipitation method are as follows: Mix the alpaca serum with an acetate buffer solution with a pH of 4-6 at a volume ratio of 1:(2-3), add 3-5% caprylic acid, mix at room temperature, then let it stand overnight at 2-8°C to fully precipitate; Centrifuge to obtain the supernatant, add an equal volume of saturated ammonium sulfate solution, mix well, let it stand overnight at 2-8°C, centrifuge to obtain the precipitate, and add 3-5 times the weight of the precipitate of saturated ammonium sulfate solution to extract 3-5 times. The last precipitate is dissolved in PBS buffer solution, and after dialysis, the pretreatment solution can be obtained; Step S2 specifically includes the following steps: S21. Loading: First, equilibrate the protein A column with 10-12 column volumes of PBS buffer solution. Then, load the pretreatment solution obtained in step S1 into the protein A column, and wash it with 5-8 column volumes of 10 mM PBS buffer solution with a pH of 7.0-7.2 at a flow rate of 1-2 mL / min to obtain a primary treatment solution; S22. Elution: Continue to wash the protein A column in step S21 with 3-5 column volumes of 20 mM citric acid buffer solution with a pH of 4.0-5.0 at a flow rate of 0.5-1 mL / min, collect the eluate, and add 1 M Tris-HCl buffer solution with a pH of 8.5 to adjust the pH of the solution to 7.2-7.4, which is natural heavy chain antibody IgG2; The packing material of the anion exchange chromatography column in step S3 is selected from one of DEAE-Sepharose, Capto-DEAE, Q Sepharose, Capto-Q, and QAE Sephadex.

2. The purification method of natural antibodies derived from alpacas as claimed in claim 1, characterized in that, The alpaca serum needs to be pretreated. The specific method is as follows: Centrifuge at 7000-9000 r / min for 10-20 min, and collect the supernatant, which is the pretreated alpaca serum.

3. The purification method of natural antibodies from alpacas as claimed in claim 1, characterized in that, Before loading, it also includes the step of sample treatment, which is specifically as follows: Mix the pretreatment solution obtained in step S1 with an equal volume of 10 mM PBS buffer solution with a pH of 7.0-7.2, and then filter it through a 0.45 μm filter membrane to obtain a sample to be purified.

4. The purification method of natural antibodies derived from alpacas as claimed in claim 1, characterized in that, Step S3 specifically includes the following steps: S31. Sample loading: First, equilibrate the anion exchange chromatography column with 8 - 10 column volumes of PBS buffer. Then, load the primary treatment solution obtained in step S2 onto the anion exchange chromatography column, and wash it with 4 - 6 column volumes of 10 mM PBS buffer with a pH of 7.0 - 7.2 at a flow rate of 1 - 2 mL / min to obtain an intermediate treatment solution. S32. Elution: Continuously wash the anion exchange chromatography column in step S31 with 3 - 6 column volumes of 50 mM Tris - HCl buffer (containing 150 - 300 mM NaCl) with a pH of 7.2 - 8.0 at a flow rate of 0.5 - 0.8 mL / min. Collect the eluate and add 1 M Tris - HCl buffer with a pH of 8.5 to adjust the solution pH to 7.2 - 7.4, which is the natural antibody IgG1.

5. The purification method of the natural antibody derived from alpacas as claimed in claim 1, wherein In step S4, the hydroxyapatite chromatography column is a ceramic hydroxyapatite chromatography column (CHT Type Ⅱ).

6. The purification method of natural antibodies derived from alpacas as claimed in claim 1, characterized in that, Step S4 specifically includes the following steps: S41. Sample loading: First, equilibrate the hydroxyapatite chromatography column with 6 - 8 column volumes of PBS buffer. Then, load the intermediate treatment solution obtained in step S3 onto the hydroxyapatite chromatography column, and wash it with 4 - 6 column volumes of 10 mM PBS buffer with a pH of 7.0 - 7.2 at a flow rate of 1 - 2 mL / min. Collect the effluent and add 1 M Tris - HCl buffer with a pH of 8.5 to adjust the solution pH to 7.2 - 7.4, which is a small amount of natural heavy - chain antibody IgG2. S42. Elution: Continuously wash the anion exchange chromatography column in step S41 with 4 - 6 column volumes of 100 mM glycine buffer with a pH of 2.0 - 3.0 at a flow rate of 0.3 - 0.5 mL / min. Collect the eluate and add 1 M Tris - HCl buffer with a pH of 8.5 to adjust the solution pH to 7.2 - 7.4, which is the natural heavy - chain antibody IgG3.

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